There is unambiguous evidence that an initial step in the development of anogenital neoplasia is infection with high risk human papilomaviruses (HPV), however it is also clear that an interplay of viral, host and environmental factors determine which infections will progress to malignancy. The finding that a subset of women develop more than one anogenital cancer provides an opportunity to focus on a group where the affect of the risk factors may be the most striking. The goal of this project is to investigate the viral and immunologic factors that contribute to single and multiple anogenital malignancies. There are 3 specific aims: 1) to determine whether specific HPV types are more frequently associated with multiple tumors, and to determine whether non-prototype-like (NPL) variants, that have been shown to be a risk factor for development of precursor lesions, are more prevalent in cases of multiple or single anogenital cancers. Characterizing the viral types and variants will, in combination with integration site studies distinguish multiple primary anogenital tumors vs. recurrence. 2) Serum antibodies to HPV VLPs are currently the best marker of the host immune response to HPV infection, and their generation is the goal of prophylactic vaccines. We propose to characterize the risk factors associated with seropositivity to HPV capsid proteins, to map the epitopes with epitope-variants; and 3) to determine whether the hot HLA genotype plays a role in progression to single or multiple malignancies. Analysis of the class II alleles in a case control series of squamous cell carcinoma of the cervix found significant associations for BRB1*1001 and DRB1*1101. Striking associations for those alleles plus DRB1*04 and DRB1*07 were seen for cases with HPV 16 tumors, and the associations were not related simply to whether the same associations are seen in other HPV-associated malignancies such as vulvar and adenocarcinoma of the cervix. The latter will also allow us to determine whether HPV 18-tumors are associated with different alleles. Finally, we will analyze the class I genes in the invasive SCC cervix groups. The combination of data on the HPV DNA type and variant in tumor tissue, the serologic profiles and the HLA genotypes should help clarify virologic and immunologic factors that influence the progression of HPV associated malignancies.